PT93951A - PREPARATION PROCESS OF IMMUNOGENIC POLYPEPTIDES AND A VACCINE UNDERSTANDING THESE POLIPEPTIDES - Google Patents

Description

70 893 SBC CASE 14445

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DESCRIPTIVE MEMORY

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the process of preparing vaccines against parasite infection of the genus Plasmodium and more particularly to the process of preparing polypeptides useful as therapeutic agents for inhibiting infection by parasites of the genus Plasmodium. malaria, which polypeptides comprise immunogenic determinants of regions of a Plasmodium surface protein flanking a central repeating domain and less than all of the repeating immunogenic repeating domain determinants; to processes for purifying these polypeptides; to expression vectors encoding these polypeptides; and to methods for the treatment of humans against malaria infection. 2. Description of Related Art Malaria is a serious, widely spread disease for which, despite years of extensive efforts, a vaccine has not yet been developed. See, for example, Science. Volume 226, page 679 (November 9, 1984). Experimentally, mammals, including humans, have been protected against infection by the etiologic agent of malaria, Plasmodium. by vaccination with irradiated sporozoites. Clyde et al., Am. J. Troo. Med. Hvg. Volume 24, p. 397 (1975) and Rieckman et al. . Bull. WHO. Volume 57 (Supp.1), p. 261 (1979). Yoshida et al. . Science. Volume 207, p. 71 (1980) report that this protection is at least partially mediated by an antibody directed against a protein on the surface of the sporozoite, the circumresporozoite (CS) protein; the monoclonal antibodies raised against the CS proteins neutralize infectivity in vitro and protect the animals in vivo. The CS protein appears to be highly, evolutionarily, conserved for the same species, but is quite varied among species. Four species of Plasmodium are known to infect man. They are E · faloiparum, E. vivax. the E-ovale and the E ·

70 893 SBC CASE 14445 malariae. the latter two occurring at a much lower frequency. Other species of scientific interest are E. berghei and E. knowlesi. being the hosts of these species, respectively, the rodents and the monkeys.

Kemp et al., WO 84-02917-A, disclose the cloning and expression of E. falciparum cDNA in E. coli.

Dame et al., Science, Vol. 225, p. 593 (1984), disclose the cloning and expression of the E. falciparum CS protein in E.Glyi. The protein is described to comprise about 412 amino acids with an approximate molecular weight of 44,000. It comprises 41 tandem repeats of a tetrapeptide. Peptides from synthetic 7-, 11- and 15- residues, derived from the repeat region, bind to monoclonal antibodies raised against the CS protein.

Antisporozoite vaccines based on the E. falciparum CS protein repeat tetrapeptides were not successful, conferring immunity in a few subjects, and this immunity was of short duration. Science 241: 522 (1988).

Consequently, the need for an effective vaccine against the malaria parasite remains unmet.

SUMMARY OF THE INVENTION The present invention generally relates to a polypeptide comprising one or more immunogenic determinants of a first region flanking a central repeat domain of a Plasmodium surface protein, one or more immunogenic determinants of a second region flanking the repeat domain, and less than all or none of the immunogenic determinants of the central repeat domain.

In one embodiment, the present invention relates to a polypeptide comprising at least one but less than all of the immunogenic repeating determinants of a Plasmodium surface protein repeat domain and one or more immunogenic determinants of regions of a protein of Plasmodium flanking the repeat domain.

70 893 SBC CASE 14445 -4

In another embodiment of the invention, the polypeptide substantially comprises all of the immunogenic determinants of the regions flanking the central repeat domain, and is devoid of immunogenic determinants of the central repeat domain. Alternatively, the polypeptide comprising substantially all of the immunogenic determinants of the flanking regions, further comprises at least one but less than all of the immunogenic determinants of the central repeat domain.

Immunogenic determinants useful in the polypeptides of the present invention preferably include those present on the surface proteins of Plasmodium falciparum. E .. vivax. L. ma lara and E. or va.

In yet another embodiment of the invention, the polypeptide is genetically fused to a carrier protein, preferably a carrier protein that improves either the expression of the polypeptide or the immunogenicity of the polypeptide, or both.

In a preferred embodiment, the polypeptides of the present invention comprise an immunogenic carrier protein, for example, the N-terminal 81 amino acids of the influenza virus non-structural protein (NS1), linked through a synthetic linker to a first region of flanking a Plasmodium ciraro-sporozoite (CS) protein which is itself fused to a second flanking region of the CS protein.

These polypeptides may additionally comprise more than one but less than all of the immunogenic determinants of the central repeat domain of the CS protein, for example, the immunogenic determinant of the repeat domain comprising a tetrapeptide having the amino acid sequence ( Asn-XY-Pro), wherein X is Ala or Val and Y is Asn or Asp. The immunogenic determinants of the central repeat domain may be positioned, for example, between the carrier protein and the first flanking region, or between the

70 893 SBC CASE 14445 first flanking region and the second flanking region.

Another aspect of the present invention includes expression vectors encoding the polypeptides, vaccines comprising the polypeptides; processes for the purification of polypeptides; and methods for the treatment of humans against malaria infection using the peptides.

Other aspects and advantages of the present invention are set forth in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 (a) and 1 (b) show ELISA results, graphically, demonstrating the antibody response of two groups of C3H / HEN mice, one NSlgβRlfA9 immunized group and the other group immunized with R32tetg2;

Figures 2 (a) and 2 (b) show ELISA results, graphically, demonstrating the antibody response of two groups of C57BL / 6 mice, one NSlgjRLf & 9 immunized group and the other group immunized with R32tet32; and

Figures 3 (a) and 3 (b) show ELISA results, graphically, demonstrating the antibody response of two groups of BALB / C mice, one NSlgβRlfA9 immunized group and the other group immunized with R32tet32. DETAILED DESCRIPTION OF THE INVENTION COHCRE.T.IZACÕES PREFERIMS. The protozoan malaria parasite, Plasmodium. has a number of proteins, specific for certain states, present on its outer cell wall. These surface proteins have been found to have three common regions, namely, a central repeated epitope domain or region and matched flanking regions. The first flanking region of the pair is fused to the carboxyl terminus of the central repeat domain and the second flanking region of the pair is fused to the amino terminus of the central repeat domain.

The polypeptides of the present invention are derived from a portion of a Plasmodium surface protein containing, less than all or none of the immunogenic determinants of the central repeat domain, between the first and second flanking region, and are expressed in amounts sufficient for use as therapeutic agents to inhibit infection by malaria parasites.

In other words, the polypeptides of the present invention have fewer tandem repeats between the first and second flanking regions than are present in the wild-type repeat regions. Thus, in the case of a polypeptide for the protection of a human against E. faloiparum infection. the polypeptide may comprise the entire first flanking region, i.e. the N-terminal flanking region of the E. faloiparum circumsporozoite protein (PfCSP), the entire second flanking region i.e. the flanking region C- terminus of the PfCSP and, between the first and second flanking regions, less than 41 tandem repeats of the PfCSP, i.e., less than 41 tetrapeptides of the formula Asn-XY-Pro, wherein X is Ala or Val and Y is Asp or Asn.

In a wild-type Plasmodium surface protein, the repeat region is immunodominant. In the polypeptides of the present invention, the number and positioning of the tandem repeats, or the repeat units, are selected so as not to mask the immuno-response to the first and second flanking regions. Preferably, the number of replicates in the polypeptides of the invention is no more than half the number of replicates present in the wild-type protein. More preferably, the number of replicates in the polypeptides of the invention is no more than about a quarter of the number of replicates present in the wild-type protein. Four species of Plasmodium are known to infect humans, with E. faloiparum followed by E. vivax and, to a lesser extent, E. malariae and E. ovale being the most prevalent. The central repeating domain of the sporozoite (CS) circulating protein in the sporozoite phase of Plasmodium-7-70-893 SBC CASE 14445 falciparum. is comprised of 41 tandem repeat tetrapeptides, thirty-one of which have the sequence (Asparagine (Asn) -Alanine (Ala) -Asn-Proline (Pro)) and four of which have the sequence (Asn-Valine ) -Aspartate (Asp) -Pro). On both sides of the central repeat domain are the so-called flanking regions containing Region I and Region II, two nearly identical CS protein regions in amino acid sequence, corresponding regions of the CS protein of E A number of E. falciparum surface proteins in the blood phase also have repeated epitopes, for example, the antigen S (Pro. -Ala-Lys-Ala-Ser-Gln-Gly-Gly-Leu-Glu-Asp), the antigen RESA (Glu-Glu-Asn-Val-Glu-His-Asp-Ala), FIRA antigen -Thr-Gln-Glu-Pro) and the antigen PF-11 (Glu-Glu-Val-Val-Glu-Glu-Val-Val-Pro) E. vivax cireum sporozoite protein contains the epitope (Asn-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala) and the P. malariae circumtesporozoite protein contains the repeated epitope (Asn-Asp-Ala-Gly) and (Asn- Ala-Gly).

The polypeptides of the present invention comprise one or more immunogenic determinants of a first region flanking the central repeat domain of a Plasmodium surface protein. one or more immunogenic determinants of a second region flanking the repeat domain and less than all or none of the immunogenic repeating determinants of the central repeat domain.

Immunogenic determinants are amino acid sequences that induce a B cell or T cell response. The immunogenic determinants generally comprise at least 6 amino acids. The precise identification of immunogenic determinants in a protein can be accomplished by conventional techniques involving localization with monoclonal antibodies and / or deletion of amino acids followed by assays of activity. Preferably, the polypeptides of the invention comprise, at least about 15 amino acids of each of the first and second flanking regions; more preferably at least about 30; and most preferably, the first and second integer flanking regions, minus the signal sequence.

In one embodiment, the polypeptides of the present invention comprise at least one but less than all of the immunogenic determinants of a central repeat domain of the Plasmodium surface protein and one or more immunogenic determinants of the surface protein regions flanking the domain of repetition.

In another embodiment, the polypeptides of the present invention substantially comprise all of the immunogenic determinants of the regions flanking the central repeat domain and are devoid of immunogenic determinants of the central repeat domain or alternatively contain at least one but less than immunogenic determinants of the central repeat domain. By " substantially all " it is understood that the first and second flanking regions are substantially integral, minus the signal sequence; preferably, each region contains no more than about 20 amino acids, and more preferably, the first and second integer flanking regions, minus the signal sequence, are used.

Preferably, the polypeptides of the present invention are hybrid polypeptides, i.e., proteins consisting of the genetic fusion between a portion of the surface protein and a carrier protein.

More preferred are hybrid polypeptides which include a carrier protein genetically fused to a portion of the Plasmodium falciparum circosporozoite (CS) protein. containing less than the totality, or devoid of the repeating tetrapeptides constituting the central repeat domain.

Particularly preferred are hybrid polypeptides in which the carrier protein improves, not only the

Immunogenicity of the transported polypeptide, but also enhances the expression of the polypeptide by a transformant. Other desirable properties of such carrier proteins include improving the purification or the formulation of the polypeptide. Examples of such carrier proteins include the N-terminal 81 N-terminal amino acids of the non-structural influenza virus 1 protein (A / PR / 8/34) (Baez et al., Nuoleic Acids Research, 2: 5845 (1980) ), R32 ([Asn-Ala-Asn-Pro) (Asn-Val-Asp-Pro)] 2) (Young et al., Science, 228: 958 (1985)); and galk.

Specific embodiments of the types of polypeptides of the present invention exemplified herein include: NSlgÎ ± -RLfA9, a fusion polypeptide comprising 81 N-terminal amino acids of the non-structural influenza virus 1 (NSlgI) protein; the flanking region containing Region I of the E. falciparum CS protein. minus the signal sequence (18 N-terminal amino acids), and the flanking region containing the

Region II, minus their first N-terminal amino acids (RLfA 9). Fusion of NSlgi with RLfA9 is facilitated by a synthetic RNA linker sequence encoding Asp-His-Met-Leu-Thr-Asp-; the NSlgÎ ± -RLfAuth, a fusion polypeptide comprising the NS1Î ± -RLfAuth; the flanking region containing Region I of the E. falciparum CS protein. minus the signal sequence, and the entire flanking region containing Region II (RLfAuth); the NSlg-γ-NÎ ± -n-X-Y-Prolβ-RLβAuth, a fusion polypeptide comprising NSlβ; (X-Y-Pro) in which X is Ala or Val and Y is Asn or Asp and n is an integer greater than or equal to one and less than or equal to 100, preferably less than 50; and, additionally, (Asn-X-Y-Pro) is positioned between NSlg- and RLfAuth; and NSlgRlFauth + (Asn-X-Y-Pro) n, a fusion polypeptide comprising NSlg; RLfAuth; and (Asn-X-Y-Pro) in which X and Y are as defined above and n is <41; and additionally, where (Asn-X-Y-Pro) is positioned between the flanking region containing Region I of the E. falciparum CS protein and the region

70 893 SBC CASE 14445 -10-flanking containing Region II, that is, in the region previously occupied by the central repeat domain.

These polypeptides are, however, only illustrative. Based on the disclosure herein, one skilled in the art will know how to construct and test other polypeptides within the scope of the invention, for example, polypeptides comprising surface protein sequences, including the circumsporozoite protein of the various malaria parasites other than E. falciparum. polypeptides comprising more or less amino acids than surface proteins, polypeptides that are chemically modified, and polypeptides that are fused to other sequences, or to additional protein or amino acid sequences. These polypeptides are readily constructed by conventional genetic engineering and / or protein synthesis techniques, and can be tested in animal models, substantially as hereinafter described. For example, a protein of the invention may comprise amino acid sequences of the flanking regions of the surface protein, such as substantially the entire circumsporozoite protein, devoid of the central repeat domain, fused to the Hepatitis B virus surface antigen (HBsAg) in a fusion protein which can form hybrid HBsAg particles, as described in European patent application EP 278,940 published August 17, 1988.

A genetic coding sequence, for surface protein flanking regions, for tetrapeptides, for synthetic DNA binder sequences and for carrier proteins, can be readily obtained by one skilled in the art using known techniques. These include the synthesis, preferably, by reverse transcription of the messenger RNA or by direct cloning of intact genomic DNA genes. Reverse transcription of E. falciparum messenger RNA is described in Ellis et al., Nature. 302: 536 (1963). Direct cloning of intact E-falciparum genomic DNA is described in Dame et al. , (cited above). Cloning and expression of polypeptides containing repeats is described in copending application no. No. 70 893 SBC CASE 14445 07 / 256,229 filed October 11, 1988, the disclosure of which is hereby incorporated by reference.

After the cloning of whole, or a portion of Plasmodium DNA. its fragments, encoding all or a portion of the surface protein, can be prepared by known techniques.

Techniques for synthesizing DNA are well known and can be performed using commercially available DNA synthesizers.

The polypeptide coding sequences can be inserted into E-coli expression vectors. many of which are known and readily available. In carrying out the present invention in E.adi, a DNA sequence encoding the polypeptide of the present invention is operably linked to a regulatory element in a DNA vector for transformation into E. coli. Numerous gram-negative bacterial expression vectors comprising these regulatory elements are available. The regulatory element comprises a promoter which binds to RNA polymerase and transcription. Regulatable, i.e., inducible or non-repressible promoters are preferred. A variety of promoters useful for expression of heterologous polypeptides are available in E. coli. These include the trp promoter and the lambda PL promoter (e.g., U.S. Patent No. 4,578,355 and Courtney et al., Nature 313: 149 (1985)). As described in more detail below, it has been found that the coding sequences encoding the polypeptides of the present invention are particularly well expressed by the E. coli expression vector pMG-1. Advantageously, pMG-1 derivatives encoding other carrier proteins other than NS1gÎ ±, for example, R32 and galK may also be used.

In carrying out the present invention in Streptomoves. a DNA coding sequence encoding the polypeptide of the present invention is operably linked to a regulatory element in a DNA vector for Streptomyces transformation. The regulatory element comprises a promoter which binds RNA-12-β-70-893 SBC CASE 14445

flS polymerase and transcription. Preferred promoters are, i.e., inducible or non-repressible promoters. A variety of promoters useful for expression of heterologous polypeptides are available from Streptomvoes. Examples include, the galactose inducible promoter of the Streptomvces galactose operon (Fornwald, S., Proc Natl. Aoad Sci USA 84 = 2130 ¢ 1987)), the constitutive promoter of the β-galactosidase gene of 2. and the long-lived trypsin inhibitor gene of S. longisporus (European Patent Application No. 87,307), which is incorporated herein by reference in its entirety. 260.7), or a temporally regulated promoter such as that described for HB and myospora (Baum et al., J. Bacteriol 120. = 71 (1988)) The regions for transcription termination in Streptomoves are derived from the 3 'of several Streptomyces genes, for example, the termination signal at the end of the Streptomoves galactose operon or that found at the end of the neomycin-phosphotransferase gene of S. fradie (Thompson and Gray, Proc Natl. Acad Sci USA 80: 519 (1983)) The sequences for exporting proteins in Str_ep_tomyO_es include isolated from the β-galactosidase gene. lividans. of the & LEP-10 gene. lividans (European Patent Application 87 307 260.7) and the S. longisporus trypsin inhibitor gene. The gene encoding the polypeptide of the present invention is incorporated into a larger DNA molecule comprising a genetic selection marker system. The selection marker system may be any of a number of known marker systems, such that the marker gene confers a novel phenotype selectable to the transformed cell. Examples include Streptomyces drug resistance genes such as thiostrepton resistance ribosomal methylase (Thompson, tape, Gene 28:51 (1982)), neomycin phosphotransferase (Thompson, et al. the ribosomal methylase resistance of erythromycin (Thompson, et al., supra). The DNA molecule may also contain a sequence for autonomous replication in Streptomoves. such as those derived from pIJ101 (Keiser, et al., Mol. Gen. Genet. 185: 223 (1982)) or a vector derived from SLP1 (Bibb, et al., Mol. Gen. Gen. Gen. 184: 230 (1981)). The DNA molecule may also contain a marker that allows the amplification of the gene.These markers, which serve to amplify the copy number of the gene in Streptomyces, include the spectinomycin resistance gene (Hornemann , S.J., Bacteriol 169: 2360 (1987)) and the arginine auxotrophic gene (Altenbuchner, et al., Mol. Gen. Genet., 105: 134 (1984)).

When the present invention is carried out in yeast, a DNA encoding sequence encoding the polypeptides of the present invention is operably linked to a regulatory element within a DNA vector for yeast transformation. For this transformation, cloning and expression systems, any available yeast host can be used. Particular examples include yeasts of the genus Hansenula. Pichia. Kluverçmyoes, Schizosaooharomvoes. Candida and Saocharçay.ges. The preferred yeast host is Saccharomyces cerevisiae. The regulatory element comprises a promoter which binds RNA polymerase and transcription. Regulatable, i.e., inducible or non-repressible promoters are preferred. A variety of promoters useful for the expression of heterologous polypeptides in yeast are available. These include the copper-inducible metallothionine (CtJP1) gene promoter and the constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase (TDH3) and alcohol dehydrogenase (ADH) glycolytic genes. Regions for the termination of yeast transcription are derived from the 3 'terminus of various yeast genes, for example the iso-1-cytochrome C (CYC1) gene. The gene encoding the polypeptide of the present invention is incorporated into a larger DNA molecule , which comprises the genetic selection marker system. The selection marker system may be any of a number of known marker systems so that the marker gene confers a novel selectable phenotype to the transformed cell. Examples include yeast genes for biosynthetic enzymes such as phosphoribosylanthranilate isomerase (TRP1) or orotodin-5 '-phosphate decarboxylase (URA3) or heterologous drug resistance genes such as the G418 resistance gene, or the

70 893 SBC CASE 14445 -14-benomylanthranilate4somerase (TRP1) or benomyl resistance (BEN1). The DNA molecule may also contain a sequence for autonomous replication in yeast, such as the 2-micron-yeast ori ori region or a chromosomal autonomous replication region (ARS), such as ARS1, and a yeast centromere (CEN), such as CEN3, to allow autonomous replication of the plasmid. Other expression systems are still known and available. For example, a variety of insect cells and expression systems, such as the baculovirus expression system for use in the expression of heterologous proteins in Lepidoptera cells, are available for expression of heterologous proteins. When it is necessary to effect expression in eukaryotic expression systems, it may be necessary to eliminate the carboxyl-terminal attachment region of the surface protein. As an example, deletion of amino acids 392-412, the sequence encompassing the carboxyl-terminal attachment region of E. falciparum, may be required. (See U.S. Patent Application Serial No. 07 / 287,934, filed December 21, 1988, the disclosure of which is hereby incorporated by reference).

Example of another expression system is that described in U.S. Patent Application Ser. Serial No. 07 / 222,202 filed July 28, 1988, the disclosure of which is hereby incorporated by reference, which relates to a bacterial strain of Salmonella transformed with a selected heterologous gene operably linked to an E. coli promoter sequence. the transformant being capable of constitutively expressing the heterologous gene product.

The polypeptides thus expressed are isolated and purified from the producer cell culture using conventional protein isolation techniques, many of which are well known in the art. An example of a useful purification scheme comprises (1) disrupting bacterial cells, (2) clarifying cell debris, (3) separating the polypeptides of the present invention from other polypeptides present in the cell extract (4) the final purification to remove residual contaminants, including, polypeptides, carbohydrates, nucleic acids, lipopolysaccharides and residual endotoxins.

In the vaccine of the present invention, an aqueous solution of the polypeptide, preferably buffered at physiological pH, may be used directly. Alternatively, the polypeptides may be admixed or absorbed with any of a number of known adjuvants. Such adjuvants include, for example, aluminum hydroxide, muramyl dipeptide and soaps such as Quil A. As a further example, the polypeptide may be encapsulated within microparticles such as liposomes. In yet another alternative, the polypeptides of the present invention may be conjugated to an immunostimulatory macromolecule such as dead Bordetella or a tetanus toxoid.

Vaccine preparations are generally described in New

Trends_and Developments_in Vaccines. Voller. al., Eds.,

University Park Press, Baltimore, MD, U.S.A. (1978). Encapsulation in liposomes is described, for example, in U.S. Pat. 4,235,877 to Fullerton. Conjugation of proteins with macromolecules is described in U.S. Pat. No. 4,372,945 to Likhite and U.S. Pat. 4,474,757 to Armor itself. al. The use of Quil A is described, for example, by Dalsgaard et al., Acta Vet. Scand. . 15: 349 (1977). The amount of polypeptide present in each vaccine dose is the amount that induces an immunoprotective response without significant adverse side effects. These amounts will vary according to the specific polypeptide used and will depend on whether or not the vaccine is used with an adjuvant. Generally, each dose will be expected to comprise from 1 to 1000æg of polypeptide, preferably from 10 to 200æg. An optimal amount of a particular vaccine can be determined by conventional studies involving the observation of antibody titers and other responses in subjects. After an initial vaccination, preferably the subjects will receive a booster about four weeks later, followed by additional boosters every six months for the duration of the risk of infection. -16- 70 893 SBC CASE 14445

Generally, intramuscular, subcutaneous or intravenous administrations are preferred, although in some cases other routes may be useful. For example, when using recombinant Salmonella, the preferred route of administration may be oral.

The following Examples are illustrative and not limiting of the invention. The sequence encoding the CS protein was provided by James Weber, Walter Reed Army Institute for Research, as a 2337 bp EcoR I fragment of XmPF1 (Dame et al., Science 225: 593 (1984)). in the EcoR I site of pUC8, a standard E. coli cloning vector (available, for example, from Bethesda Research Laboratories, Inc., Gaithersburg, MD, USA). The resulting pUC8 derivative is designated clone 1 of pUC8. EXEMP.

Construction of pNSlQ1RLfA9

Briefly, briefly, the construction of pNSl81RLfA9 was performed as follows. A first aliquot of pCSP (described below) was digested with Fok I restriction endonuclease, an E. coli expression vector containing a 1216 bp fragment encoding all but the first 18 amino acids of the circum- sporozoite (CS) of P. falciparum, the ends thereof were filled (Klenow fragment) and digested with BamH I restriction endonuclease. The resulting 318 bp fragment encoding amino acids 19 (Leu) to 123 (Pro) CS protein.

A second aliquot of pCSP was digested with TlIII restriction endonuclease, its ends filled, and digested with restriction endonuclease Sal I. The resulting 655 base pair fragment encoding amino acids 297 (Gly) to 412 (Asn) of the CS protein was recovered by electroelution. (9 N-terminal amino acids, 288 (Pro) at 296 (Gin), of the flanking region containing Region II are lacking at this time.

The 318 base pair and 655 base pair CS protein gene fragments were ligated into the E. coli pUC18 expression vector (described below) previously digested with the

70 893 SBC CASE 14445 restriction endonucleases BamH I and Sal I. The resulting vector was designated pUCRLfA9. PUCRLfA9 was digested with BamH I restriction endonuclease, its ends were filled and Sal I restriction endonuclease was digested. The resulting 1035 base pair fragment encoding the amino acids of the CS protein, 19 to 123 and 297 to 412, was purified by electroelution.

The pMG-1 expression vector (described below), containing a DNA fragment, encoding the N-terminal 1 (Met) to 81 (Met) amino acids of the influenza virus non-structural protein 1 was digested, and a synthetic DNA ligand sequence with the restriction endonucleases EcoR V and Xho I. The 1035 base pair fragment previously isolated from pUCRLfA9 was then ligated to pMG-1. The resulting expression vector, pNSlglRLfA9, encodes a protein having the following sequence: NSlgÎ ± -Asp-His-Met-Leu-Thr-Asp-Pro-CSgÎ ± -23-CS297_4i2

The construction of pNSl01RLfA9 is described in detail below.

A. Building the pCSP

The plasmid DNA of purified pUC8 clone 1 (40æg) was digested with restriction endonucleases Stu I and Rsa I (100 units of each enzyme) in 400æl of medium buffer (comprising 50 mM Tris, 50 mM NaCl mM, 1 mM dithiothreitol (DTT) and 10 mM MgCl2, pH 7.5) for 1.5 hours at 37 ° C. The resulting 1216 base pair fragment, encoding all but the first 18 amino acids (believed to encode the CS protein signal sequence) of the circosporozoite (CS) protein, was isolated by electrophoresis on a polyacrylamide gel (PAGE ) at 6% and recovered by electroelution.

10æg of the pAS1 expression vector (ATCC 39262, described more fully in U.S. Patent 4,578,355 to M. Rosemberg) was digested with restriction endonuclease BamH I (25 units) in 200æl of mean buffer (described above) for 1.5 hours at 37 ° C. The cut plasmid was then treated for 15 -18- 70 893 SBC CASE 14445 minutes at 25 ° C with DNA Polymerase I, Large Fragment (5 units Klenow Fragment in 20 mM Tris-HCl, pH 7.5, 7 mM MgCl 2 , 60 mM NaCl, 6 mM 2-mercaptoethanol and 0.25 mM of each of the four deoxynucleotide triphosphates to fill the BamH site end). The circumsporozoite protein fragment (1 pg) was then ligated to this vector (100 ng) in 30 pl of the ligase buffer (comprising 50 mM Tris, 1 mM DTT, 10 mM MgCl2, 100 pM rATP, pH of 7.5) with a T4 DNA ligase unit for 16 hours at 4 ° C.

The ligation mixture was used to convert. coli strain MM294CI +. Polymer-resistant colonies were obtained and investigated for insertion of the CS gene fragment into pAS1. A plasmid with the correct construction (PCSP) was identified. B.,,. U.S. Pat.

Purified pCSP plasmid DNA (100æg) was digested with restriction endonuclease Fok I (100 units) in 400æl of medium buffer (described above) for 3 hours at 37øC. Subsequently, the plasmid was treated with DNA fragment Polymerase I (described above) to fill the Fok I site at the end. The plasmid was then digested with restriction endonuclease BamH I (100 units) in 400 μl of buffer (described as a slurry) for 3 hours at 37øC. The resulting 318 bp fragment encoding amino acids 19-123 of the CS protein was isolated by electrophoresis on a 6% polyacrylamide gel (PAGE) and recovered by electroelution.

An additional aliquot (100æg) of TcIII restriction endonuclease (100 units) of pCSP was digested in 400æl of medium buffer (described above) for 3 hours at 65 ° C. Subsequently, the plasmid was treated with DNA fragment Polymerase I (described above) to fill the TIIIII site. The Sal I restriction endonuclease plasmid (100 units) was then digested in 400æl of medium buffer , during

70 893 SBC CASE 14445 3 hours at 37 ° C and the resulting 655 base pair fragment encoding amino acids 297-412 of the CS protein was isolated by electrophoresis on a 6% polyacrylamide gel and recovered by electroelution.

10æg of pUC18 expression vector (Yanisch-Perron gi al., Fiene, 22: 103 (1985)) was digested, a standard K. fili cloning vector (available, for example, from Bethesda Research Laboratories, Inc., Gaithersburg, MD), with the restriction endonucleases BamH I and Sal I (20 units each) in 200æl of buffer medium (described above) for 2 hours at 37 ° C. The 318 base pair (1æg) filled end-BamH I / Fok I fragment and 655 base pair (1æg) I-end-filled / Sal I fragment were then ligated into pUC18 in 30 ul of ligase buffer (described above), with a T4 DNA ligase unit, for 16 hours at 4 ° C. A plasmid of the correct construction (pUCRLfÎ "9) was identified. C-_Construction of pMG-1

10 μg of expression vector pMG27N- (M.Gross et al., Mol. Cell Biol., 2: 1015 (1985)) was digested with restriction endonucleases BamH I and Sac I (50 units each) in 200 μl of medium buffer (described above) for 3 h at 37 ° C.

10 Âμg of expression vector pAPR801 (Young gà © al, Proo.Hatl.Acad.So.U.S. 22: 6105 (1983) was digested containing the coding region of the non-structural protein (NS1) of influenza virus (A / PR / 8/34) (Baes, et al., Nucleic Acids Research 8: 5845 (1980)) with the restriction endonucleases Ncol and BamH I (20 units each) in 20 μl buffer (50 mM Tris, 1 mM DTT, 10 mM MgCl 2, and 100 mM NaCl, pH 7.5) for 2 hours at 37 DEG C. The resulting 230 bp fragment encoding the first 81 N-terminal amino acids of NS1 , was isolated by electrophoresis on a 6% polyacrylamide gel (PAGE), and recovered by electroelution.

40 ng of BamH Ι / Sac I cut pMG27N (described above) was ligated with 80 ng of the Nco Ι / BamH I fragment encoding

70 893 SBC CASE 14445 to NSlg2 of 230 base pairs and with 80 ng of a synthetic linker having the following sequence:

Asp HisMet Lau Thr Asp B TrT 5 'CATGGATCAT ATGTT AACAGATATCAAGGCCTGACTGACTGAGAGCT 3'

BamHI Saci 3 'CTAGTATACAATTGTCTATAGTTCCGGACTGACTGACTC 5' The resulting plasmid, pMG-1, was identified with the BamH I site of the sequence encoding NS181, bound to the BamH I site of pMG27N-; with the Nco I site, of the sequence encoding NSlg-β bound to the Nco I site of the synthetic linker; and with the Sac I site of the synthetic ligand bound to the Sac I site of pMG27N-. This vector introduces unique restriction sites to facilitate insertion of DNA fragments into any of three reading strands, results in the insertion of TGA termination codons into all three reading strands downstream of the ATG initiation codon of the binding site of ribosome cll and, when expressed, results in NSlgII fusion proteins of all three reading chains. Digestion of pMG-1 with the Nde I restriction endonuclease and subsequent vector binding, as described above, results in the expression of a non-fusion protein (i.e., non-fused to NSlgl). H._Nonstruction of pNSleiRLfA9

The pUCRLf expression vector (100 μg) was digested with restriction endonuclease BamH I (100 units) in 400 μl of high buffer (described above) for 3 hours at 37 ° C. The cut plasmid was subsequently treated with DNA-Polymerase I, Large Fragment (described above) to fill the BamH I site at the far end. The plasmid was then digested with Sal I restriction endonuclease (20 units) in 400 μl of medium buffer (described above) for 3 hours at 37 ° C, the resulting 1035 base pair fragment was isolated by electrophoresis on a 6% polyacrylamide gel (PAGE), and recovered by electrolux.

The pMG-1 expression vector (10æg) was digested with the restriction endonucleases EcoR V and Xho I (25 units each) in 400æl medium buffer (described above) for 3 hours at 37øC. The filled-end BamH I / Sal I fragment of

(400 μg) was then ligated into this vector (100 ng) in 30 μl of ligase buffer (described above) with a T4 DNA ligase unit, for 10 min. 16 hours at 4 ° C.

The ligation mixture was transformed into E. coli of strain MM294C1 +. Ampioillin resistant colonies were obtained and investigated for the presence of clones containing the appropriately targeted inserted gene. A plasmid of the correct construction (pNSl81RLfA9) was identified, transformed into E. coli of strain AR58 (CIts857) and tested for expression of the circumsporozoite protein gene product devoid of the first 18 N-terminal amino acids ( (β-β-g), the central repeat domain and the N-terminal 9 amino acids (Î »243-293) flanking reaction containing the fused II region (RLfA9) through 6 amino acids (Asp-His- Met-Leu-Thr-Asp), derived from the synthetic ligand bound to the pMG-1 expression vector, the N-terminal 81 amino acids of the non-structural influenza 1 protein, NSlg-1. The NS1 gene has the following sequence: Proline, separating Asp (from the C-terminus of the synthetic linker) from RLfA9 (CSig_i23_CS2g7_4i2) is a filled BamH I site artifact of the BamH I / Fok I fragment of pCSP.

Cells were grown in Luria-Bertani Broth (LB) at 32øC to an absorbance at 650 nm (Aggg) of 0.6 and induced by temperature at 42øC for 3 hours to initiate the transcription of PL promoter of the expression plasmid and subsequent translation of the NS1gÎ ± derivative of the CS protein. Cells were harvested in 1 ml, sedimented aliquots resuspended in lysis buffer (10 mM Tris-HCl, pH 7.8, 25% (vol / vol) glycerol, 2% 2-mercaptoethanol, 2% dodecylsulfate (SDS), 0.1% bromophenyl blue) and incubated in a heating block at 105 ° C for 5 minutes.

Proteins were separated by SDS-PAGE (13% acrylamide, acrylamide: bis-acrylamide ratio of 30: 0.8). The proteins were transferred to nitrocellulose and the NSlgiRLfA9 protein, produced in E-coli. was detected by analysis of

70 893 SBC CASE 14445

Western blotting using polyclonal antibodies reactive with a CS protein domain designated Region I (Dame et al., Science 225: 593 (1984)) as well as polyclonal antibodies reactive with NSlg-β protein. It was also found that NSlg protein RLfA 9 produced in. coli was non-reactive with a set of 5 monoclonal antibodies directed against the tetrapeptide repeat domain of the E. falciparum CS protein. EXAMPLE 2 The expression vector of E. coli. pCSP, (described above) was digested with restriction endonucleases BamH I and Fok I in medium buffer. The resulting DNA fragment, encoding the flanking region containing Region I, minus the first N-terminal 18 amino acids, was recovered by electroelution.

A second aliquot of pCSP was digested with the restriction endonucleases T thIII and Sal I in medium buffer. The resulting DNA fragment, encoding the flanking region containing Region II minus the first N-terminal 9 amino acids, was recovered by electroelution. The E. Coli expression vector pUC18 (described above) was digested with restriction endonucleases BamH I and Sal I in medium buffer.

To restore the 9 N-terminal amino acids (CS2gg_296) of the region II containing the C-terminal flank of the central repeat domain of the CS protein, which amino acids were lost in the digestion of pCSP with the restriction endonuclease Tth11 I , a synthetic DNA fragment containing a Fok I end and a T thIII end was prepared, and having the following sequence

Asp Pro Gly Asn jay s Asn Asn Gin J51y Asn Gly Gin 5 '* ATCCCG6GAATAAÁAACAACCAAGGTAATGGACA1 3'

The BamH Ι / Fok I fragment, the TthIII I / Sal I fragment and the synthetic fragment, were ligated into the BamH I-digested pUC18 and the BamHI-I fragment. Salt I.

The resulting plasmid, pUCRLfAuth, was digested with the restriction endonuclease BamH I in medium buffer, the ends filled, and digested with restriction endonuclease Sal I. The resulting DNA fragment encoding the authentic circumniosporozoite protein, which lack the first 18 N-terminal amino acids and the central repeat domain, was recovered by electroelution. The isolated end-filled BamH I / SAL I fragment was then ligated into the Euoliol expression vector encoding the NS181, pMG1 (described above), which had been previously digested with restriction endonucleases EcoR V and Xho I. The vector of the resulting expression, pNSlg RRLfAuth, expresses a protein having the sequence: NSlg-L-Asp-His-Met-Leu-Thr-Asp-Pro-CSg. 123-Gly-S288-412 (Â Glycine separating the CS flanking regions containing the

Region I and Region II (CSig_123 and cs288-412) is an artifact of the synthetic DNA ligand sequence, Fok I / Tlllll I.

The complete nucleotide and amino acid sequences are shown for NSlgγ RLfAuth:

70 803 SBC CASE 14445 -24- ^ rtS! ATggatccaaacactgtgtcaagctttcaggtagattgctttctttggcatgtccgcaaa 1 --------- * --------- + --------- + --------- ».------- - + -.-------- «60 TAcctaggtttgtgacacagttcgaaagtccatctaacgaaagaaaccgtacaggcgttt i

MetAspProAsnThrValSerSerPheGlnValAspCysPheLeuTrpHisValArgLys -cgagttgcagaccaagaactaggtgatgccccattccttgatcggcttcgccgagatcag 61 --------- + --------- + --------- + --------- + ------ --- + --------- + i20 gctcaacgtctggttcttgatccactacggggtaaggaactagccgaagcggctctagtc

ArgValAlaAspGlnGluLeuGlyAspAlaProPheLeuAspArgLeuArgArgAspGln - aaatccctaagaggaaggggcagcactcttggtctggacatcgagacagccacacgtçct I2i -------------------------- + - ---- + ------------- ------ + 180 tttagggattctccttccccgtcgtgagaaccagacctgtagctctgtcggcgtgcacga

LysSerLeuArgGlyArgGlySerThrLeuGlyLeuAspIleGluThrAlaThrArgAla -

ggaaagcagatãgtggagcggattctgaaagaagaatccgatgaggcacttaaaatgacc 181 --------- + --------- + --------- + --------- + -------- - + --------- + 240 cctttcgtctatcacctcgcctaagactttcttcttaggctactccgtgaattttactgg GlyLysGlnlleValGluArglleLeuLysGluGluSerAspGluAlaLeuLysMetThr -atdgatcatatgttaacagaqccqTTATTCCAGGAATACCAGTGCTATGGAAGTTCGTCA 241 tac 91 Me ctagtatacaattgtcta spHisMetLeuThrAsp

tA ggqjAATAAGGTCCTTATGGTCACGATACCTTCAAGCAGT PrdLeuPheGlnGluTyrGlnCysTyrGlySerSerSer 300

AACACAAGGGTTCTAAATGAATTAAATTATGATAATGCAGGCACTAATTTATATAATGAA 301 - -------- h ---------- + 360

TTGTGTTCCCAAGATTTACTTAATTTAATACTATTACGTCCGTGATTAAATATATTACTT

AsnThrArgValLeuAsnGluLeuAsnTyrAspAsnAlaGlyThrAsnLeuTyrAsnGlu - TTAGAAATGAATTATTATGGGAAACAGGAAAATTGGTATAGTCTTAAAAAAAATAGTAGA 361 --------- + --------- + --------- + --------- + ------ --- + --------- + <5 20 AATCTTTACTTAATAATACCCTTTGTCCTTTTAACCATATCAGAATTTTTTTTATCATCT LeuGluMetAsnTyrTyrGlyLysGlftGluAsnTrpTyrSerLeuLysLysAsnSerArg -

TCACTTGGAGAAAATGATGATGGAAATAATAATAATGGAGATAATGGTCGTGAAGGTAAA 421 --------- + --------- + --------- + --------- + -------- - + --------- + 480 agtgaacctcttttactactacctttattattattacctctattaccagcacttccattt

SerLeuGlyGluAsnAspAspGlyAsnAsnAsnAsnGlyAspAsnGlyArgGluGlyLys -

70 893 SBC CASE 14445 -25- 481

Regression X GATGAAGATAAAAGAGATGGAAATAACGAAGACAACGAGAAATTAAGGjAAACCAAAACAT CTACTTCTATTTTCTCTACCTTTATTGCTTCTGTTGCTCTTTAATTCC AspGluAspLysArgAspGlyAsnAsnGluAspAsnGluLysLeuArç ---------- + TTTGGTTTTGTA LysProLysHis 540

aatggacaaGGTCACAATATGCCAAATGACCCAAACCGAAATGTAGATGAAAATGCTAAT 601 --------- + --------- + --------- + --------- + -------- - + --------- + 660

ttacctgtt CC AGTGTT AT ACGGTTT ACTGGGTTTGGCTTT AC ATCT ACTTTT ACG ATT A m

AsnGlyGlnGlyHisAsnMetProAsnAspProAsnArgAsnValAspGluAsnAlaAsn -

GCCAACAATGCTGTAAAAAATAATAACCAAGAACCAAGTGATAAGCACATAGAACAA 661 --------- + --------- + --------- + --------- + -------- - + --------- + 720

CGGTTGTTACGACATTTTTTATTATTATTGCTTCTTGGTTCACTATTCGTGTATCTTGTT

AlaAsnAsnAlaValLysAsnAsnAsnAsnGluGluProSerAspLysHisIleGluGln - &gt; Region tt

TATTTAAAGAAAATAAAAAATTCTATTTCAlACTGAATGGTCCCCATGTAGTGTAACTTGT '80 721 541 399 AAAAAATTAAAGCAACCAGGGGATGGTAATCCT OATCCc T-W aataaaaacaaccaaggt TTTTTTAATTTCGTTGGTCCCCTACCATTAGGA IA 'ui LysLysLeuLysGlnProGlyAspGlyAsnPro CTAGGg JAA AspPrc CCC Gly ttatttttgttggLtcca m AsnLysAsnAsnGlnGly

ATAAATTTCTTTTATTTTTTAAGATAAAG1TGACTTACCAGGGGTACATCACATTGAACA

TyrLeuLysLysIleLysAsnSerlleSer

HM

ThrGluTrpSerProCysSerValThrCys -

GGAAATGGT

\ TTCAAGTTAGAATAAAGCCTGGCTCTGCTAATAAACCTAAAGACGAATTA 781 CCTTTACCA SSI 840 rAAGTTCAATCTTATTTCGGACCGAGACGATTATTTGGATTTCTGCTTAAT GlyAsnGlyJIleGlnValArglleLysProGlySerAlaAsnLysProLysAspGluLeu -

GATTATGAAAATGATATTGAAAAAAAAATTTGTAAAATGGAAAAATGTTCCAGTGTGTTT 841 --------- + --------- + --------- + --------- + -------- 900

TABLE

AspTyrGluAsnAspIleGluLysLysIleCysLysMetGluLysCysSerSerValPhe -AATGTCGTAAATAGTTCAATAGGATTAATAATGGTATTATCCTTCTTGTTCCTTAATrAG 901

A A TTACAGCATTTATCAAGTTATCCTAATTATTACCATAATAGGAAGAACAAGGAATT AsnValValAsnSerSerIleGlyLeuIleMetValLeuSerPheLeuPheLeuAsn rc End

ATAA 961 ---- 964

TATT 960

70893 SBC CASE 14445 EXEMELP. .The

Construction of pNSlglRLfAuth + CNANP) n

The pNSlgβRLβAuth (described above) was digested with the Sma I restriction endonuclease in medium buffer (described above) and KClâ, ") containing at 25 ° C for 3 hours.

A synthetic DNA linker having the sequence:

AsnAlaAsnProAsnAlaAsnPro 5 'AACGCAAACCCAAATGCAAACCCC 3' 3 'TTGCCTTTGGGTTTACGTTTGGGG 5' on Sma I digested pNSl8: 1RLfAuth. Synthetic DNA ligand encodes (NANP) 2 (single letter symbols designating amino acids, N = Asparagine (Asn); A = alanine ( Ala), and P = proline (Pro)), the tetrapeptide constituting the so-called consensus sequence of the immunodominant repeat domain of the CS protein. The digestion of pNSl81RLfAuth with restriction endonuclease Sma I allows the binding of any number of repeated tetrapeptides, encoded by a synthetic DNA ligand, into the region of the CS protein initially occupied by the immunodominant repeat domain. In this way, additional DNA fragments encoding the tetrapeptide can be ligated into the vector. The resulting plasmid, pNSlgβRLfAuth + (NANP) 2 encodes a protein having the sequence: NSlgÎ ± -Asp-His-Met-Leu-Thr-Asp-Pro-CSÎ " and EXAMPLE 4

The pUCRLfAuth expression vector (described above) was digested with BamH I restriction endonuclease. To the BamH I digested pUCRLf, a synthetic DNA fragment encoding (NANP) 4 was ligated. The synthetic DNA fragment had the following sequence:

Pro Asn Ala Aai Pro Asn Ala Asn Pro Asn Ala Asn Bro Asn Ala Asn Pro 5 'GATCCCAATGCAAACCCAAATGCAAACCCAAACGCTAACCCCAACGCTAACCCC 3' 3 'GGTTACGTTTGGGTTTACGTTTGGGTTTGCGATTGGGGTTGCGATTGGGGCTAG 5'

70 893 SBC CASE 14445 The resulting plasmid was designated pUC18 &lt; NANP) 4RLfAuth. The expression construct pUC18 (NANP) 4RL Auth was digested with end-filled BamH I restriction endonuclease (Klenow fragment), digested with Sal I restriction endonuclease, and the resulting DNA base pair fragment was recovered by electroelution .

The BamHI-end-filled fragment / Sal I was ligated into the pMG1 expression vector, encoding the NS181 (described above), previously digested with restriction endonucleases EcoR V and XhoI. The resulting plasmid was designated pNSI- NANP ^ RLfAuth and encodes a protein where the repeating tetrapeptides encoded by the synthetic DNA fragment are inserted between the amino acid 81 (Met) of NS181 and the N-terminal Asp of the synthetic DNA ligand Nco I / Sac I NSl- (NANP) 4-Asp-His-Met-Leu-Thr-Asp-Pro-CSg 3-3 288-412 ΕΧΕΜΕΙιΩ-5-

The construct of pNS184 (NVDP) 4RLfAuth was performed as described above for pNS188 (NANP) 4RLfAuth with the exception of the synthetic DNA ligand, encoding (NVDP) 4 ( the variant tetrapeptide sequence of the central repeat domain of the CS protein), have the following sequence:

The resulting plasmid encodes a protein having the sequence: NSl81 (NVDP) 4-Asp-His-Met-Leu-Thr-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp- Asp-Pro-CS19-123-Gly-CS288_412 EXEHPLP-6.

Isolation of NS1βRLβ9 from E. coli

After induction of the synthesis of NSl81RLfA9 on a temperature sensitive lysogen 28-70-893 SBC CASE 14445 (CIts857), the bacterial cells were collected by centrifugation and the resulting pellet was frozen at -70 ° C. Approximately 12 g of the frozen and concentrated cells were thawed by diluting in 120 ml of a lysis buffer solution (pH8) containing 50 mM Tris (hydroxymethyl) aminomethane (TRIS), 10 mM ethylenediaminetetraacetic acid (EDTA), 5 % glycerol and 10 mM dithiothreitol (DTT). Lysozyme (Sigma Chemical Co., St. Louis, MO, U.S.A.) was added to a final concentration of 0.2 mg / mL of diluted cells and the solution was shaken at 4 ° C for 30 minutes. Cells were sonicated using a Branson sonicator until the solution appeared to be liquefied. A 10% deoxycholate solution was added to a final concentration of 0.1% (v / v) and the solution was centrifuged at 15,000 x G for 30 minutes at 40 ° C in a Sorvall RC 5B centrifuge (Dupont).

The supernatant was discarded and the remaining protein-containing pellet was suspended in 100 ml of a buffer solution (pH 10) containing 50 mM glycine, 2 mM EDTA and 5% glycerol. The suspension was sonicated as described above and Triton® X-100 (Sigma) was added to a final concentration of 1% (v / v). The sonicated solution was stirred at 4 ° C for 30 minutes and centrifuged as described above.

To the supernatant containing protein, urea was added to a final concentration in 8 M urea and the sample was titrated to pH 5.5 with a 50% solution of acetic acid. The sample was chromatographed on a 25 ml QAE Sepharose® Fast Flow (Pharmacia) column pre-equilibrated in a buffer solution (pH 5.5) containing 20 mM sodium acetate, 1% Triton X-100 and 8M urea, flow rate of 300 cm / h. The protein was in the unbound fraction and applied to a 10 ml SP Sepharose® Fast Flow (Pharmacia) column pre-equilibrated in a buffer solution (pH 5.5) containing 20 mM sodium acetate and 8 M urea, with a flow rate of 120 cm / h. The effluent was monitored for absorbance at 280 nm. The protein was eluted from this column with a buffer (p &amp; 8) containing 100 mM Tris and 8 M urea. SDS-PAGE analysis of the resulting product revealed a SBC CASE 14445 the main band with an apparent Mr of 40,000 kD and a purity of approximately 80%. From the purification process 12 mg of protein containing approximately 14 endotoxin units / mg protein were obtained. EXAMPLE 1

Isolation of NSlslRLfA9 from E. coli.

After induction of the NS181RLfA9 synthesis in a temperature sensitive lambda lysogen, the bacterial cells were harvested by centrifugation and the resulting pellet was frozen at -70 ° C. Approximately 636 g of the frozen and concentrated cells were thawed by diluting to 2200 ml of a buffer solution (pH 8.0) containing 60 mM Tris, 12 mM EDTA, 6% glycerol and 12 mM dithiothreitol (DTT) . The thawed cells were passed through a Manton Gaulin homogenizer twice, at 41369--48264 kPa. A 10% deoxycholate solution was added to a final concentration of 0.1% (v / v), the lysate was stirred at 4 ° C for 30 minutes and centrifuged at 10,000 x G in a Sorvai RC centrifuge 5B (Dupont) at 4 ° C for 60 minutes.

The pellet was discarded and each ml of protein-containing supernatant was added 25 ul of Biocril 1050 or 2100 bead mixture (SUPELCO). The solution was stirred and centrifuged as described above.

The pellet was discarded and the remaining supernatant containing protein was added solid ammonium sulfate over a period of five minutes to 20% saturation.

The sample was stirred and centrifuged as described above. The pellet was suspended in 400 ml of a buffer solution (pH 5.5) containing 20 mM sodium acetate, 1% Triton X-100 and 8M urea. This suspension was centrifuged and the supernatant dialyzed against a buffer solution (pH 8.0) containing 100 mM Tris and 8 M urea. The retentate was adjusted to pH 5.5 and chromatographed on a 100 ml column of SP Sepharose Fast Flow (Pharmacia) previously equilibrated with a buffer solution (pH 5.5) containing 20 mM sodium acetate, 1% Triton X-100 and urea at a flow rate of 10 ml / minute. The column was washed with equilibration buffer, followed by a buffer containing 0.1 M Tris and 8 H urea at pH 8. The column was eluted with 500 ml of a linear gradient of sodium chloride, 0.0 to 0.5 M, prepared in a buffer (pH 8) containing 0.1 M Tris and 8 M urea. The protein was eluted with 0.3 M sodium chloride. fractions containing the product were pooled and concentrated in an Amicon stirred cell using a YM 10 membrane and dialyzed against a buffer solution (pH 8.0) containing 20 mM Tris, followed by dialysis against a phosphate buffered saline solution (pH 7, 0). SDS-PAGE analysis of the resulting product revealed a major band with an apparent Mr of 40,000 kD and a series of minor components. From the purification process 25 mg of protein containing 144 endotoxin units / mg protein were obtained. EXAMPLE 8

Isolation of .RlgCSP from,. E.sali 0 R16CSP, expressed in EL. coli. was prepared by ligating an isolated Xho II fragment from pUC8 clone 1 (described above) into the previously described BCHH restriction endonuclease pCSP (described above). The R16CSP used as the ELISA capture antigen in the following Example 9 was purified as below.

After induction of the synthesis of R16CSP in IL. coli. the bacterial cells were collected by centrifugation and the resulting pellet was frozen at -20 ° C. Approximately 373 g of concentrated and frozen cells were thawed in 1.43 l of buffer (pH 3.0) containing 50 mM Tris, 10 mM EDTA, 5% glycerol and 10 mM dithiothreitol. A solution of 10% (w / v) deoxycholate to a final concentration of 0.1% (v / v), after which cells were passed through a Manton-Gaulin homogenizer at 48 264 kPa twice. To the homogenate was added a 10% (w / v) solution of polyethyleneimine (BRL) in 0.5 M Tris, pH 8.0 buffer, to a final concentration of 0.5%. The solution was stirred at 4 ° C for 1 hour and centrifuged at 13,000 x g in a Sorvall RC 2B (Dupont) centrifuge at 4 ° C for 30 minutes at 70-83 ° C CASE 14445.

The pellet was discarded and to the supernatant solid ammonium sulfate was added to a saturation of 35% for 5 minutes. The solution was stirred and centrifuged as previously described.

The pellet was suspended in a buffer (pH 8.0) containing 300 ml of 20 mM Tris and 10 mM EDTA. A solution of 300 ml of 8 M urea, 2.7 l of 10 mM sodium acetate and 4 M urea was added, and the pH was adjusted immediately to 4.0. The sample was centrifuged as described above.

The supernatant was applied to a 50 ml SP-Sepharose®, Fast Flow (Pharmacia) column equilibrated in a buffer (pH 4.0) containing 20 mM sodium acetate and 4 M urea. The column was washed with buffer (pH 5.0) containing 20 mM sodium acetate and eluted with 250 ml of a linear gradient of sodium chloride, 0.0 to 1.0 M, in wash buffer. The product was eluted with approximately 0.3 M sodium chloride.

A 50 ml aliquot of the ion exchange product was adjusted to pH 2.3 with 10% (v / v) trifluoroacetic acid (TFA) and a C4 reverse phase column (Vydac, 1 X 25 cm) was chromatographed, equilibrated in 0.1% TFA. A linear gradient of 0 to 60% acetonitrile (ACN) in 0.1% TFA was applied over 45 minutes, and the product was eluted with approximately 60% acetonitrile. The reverse phase product was neutralized by the addition of 25 μg / ml of a 1M solution of ammonium bicarbonate.

Approximately 45 ml of the reverse phase product was dialyzed against a buffer (pH 5.0) containing 2M guanidine hydrochloride, and concentrated to 20 ml on a YM 30 (Amicon) membrane. A 10 ml aliquot was chromatated on a 2.5 x 50 cm column of Superose® 12 (Pharmacia) equilibrated in a buffer (pH 5.0) containing 2.0 Âμ guanidine hydrochloride. Protein which was eluted with an apparent Mr of 358 kD was dialyzed against a buffer (pH 4.5) containing 20 mM sodium acetate. Coomassie stained SDS-PAGE analysis of the Superose product revealed two major bands with an apparent Mr of 72

70 893 SBC CASE 14445 kD and 70 kD. The N-terminal amino acid analysis and assay of the final product agreed, at less than 15%, to the expected values. EXAMPLE 9

ResP.Qstas. of AnticorRjos. MSl91RLfA9

To evaluate their immunogenicity, purified NSlg-RLfA9 antigen was inoculated into three mouse strains, C57BL / 6 (H-2b); BALB / C (H-2d); and C3H / HEN (H-211). It has previously been shown that only H-2 haplotype mice produce antibodies against the repetitive epitope of the EL circumnosporozoite protein. falciparum. The antigen was injected with Freund's adjuvant and the serum samples from immunized animals were analyzed in an enzyme-linked immunosorbent assay (ELISA). Control animals were immunized with R32tet32, an antigen containing repeating tetrapeptides of the CS protein. Inoculation of all three mouse strains with NS11gRLfA9 resulted in the production of antibodies reactive with the non-repeating (flanking) region of the circosporozoite protein. The details and results of the immunogenicity assay were presented below.

Each of the three mouse strains was separated into two groups, each group comprising four to five animals. The first group of animals was immunized with NSlβRLfA9 and the second group was immunized with R32tet32 (J. Young et al., Science, 228: 958 (1985)). The R32tet32 contains two Xho II fragments, each fragment encoding a peptide having the sequence [(Asn-Ala-Asn-Pro) (Asn-Val-Asp-Pro)] 20 tet32 is a 32 amino acid peptide encoded by the gene of tetracycline resistance, read out of the chain (&quot; read out of frame &quot;).

The NSl31RLfA9 antigen, purified according to Example 6, was mixed with complete Freund's adjuvant immediately prior to administration. The mice (6 to 8 weeks of age) were immunized subcutaneously each with 50 pg of antigen administered at a dose of 200 μg in the right hindquarter. The mice were immunized twice, the first time with a

Single dose of 200 μl containing 50 μg of antigen in complete Freund's adjuvant. Booster injections were administered four weeks later according to the same protocol that was used for the first injections except that the antigen was emulsified in incomplete Freund's adjuvant.

The animals were bled 7 days after the second immunization. All blood from all animals was pooled into one group, coagulated overnight at 4 ° C and centrifuged to separate the serum, which was then stored at -70 ° C. An ELISA was used to test the pooled sera for the presence of antibody produced against R32tet32, NS81RLfA9 or R16CSP. (R16CSP was prepared and purified as described above). A &quot; sandwich &quot; ELISA incorporated R32tet32, R16CSP and NSlgβRLÎ ± as capture antigens adsorbed onto the walls of the well of a microtiter plate. The capture antigen was adsorbed onto the well walls of a microtiter plate by adding 50 μl of a PBS solution containing 0.75 μg of capture antigen and 0.2 μg of boiled casein to the well. This solution was prepared by adding 8 μl (3.76 μg) of capture antigen to 2.5 ml of a solution consisting of 4 μl of a boiled 5% casein solution (described below) in 5 ml of brine (PBS) (an aqueous solution comprising 0.8% NaCl, 0.217% Na 2 HPO 4 .7H 2 O, 0.02% KH 2 PO 4, and 0.02% KCl, with a pH of 7, 4).

After incubation overnight at room temperature, the contents of the wells were aspirated and the remaining active sites on the plates were blocked with a boiled 5% casein solution (5 g / 1 of Casein (JT Baker Chemical Co.), 0.1 g / l Thimersol (Sigma Chemical Co.), 0.02 g / l phenol red (Sigma Chemical Co.), 900 ml PBS, pH 7, 4, and 100 ml of 0.1N NaOH) (99: 1) with 1% Tween 20 (polyoxyethylene sorbitan monolaurate, Sigma Chemical Co.).

Samples of mouse sera were diluted 1: 100; 1: 1000; 1: 10,000; 1: 100,000 and 1: 1,000,000 in a 0.5% CASE 14445 caseinate solution containing 0.025% Tween 20. CASS 14445 CASS 14445 casein solution. To each well was then added the test serum and after 2 hours of incubation, the serum was removed and the wells were washed twice with a PBS solution containing 0.05% Tween 20. After addition of a peroxidase-conjugated anti-mouse IgG Ab , diluted 1: 2000, with the same diluent used for the sera. After one hour of incubation, the contents of the wells were aspirated, washed 3 times with the PBS solution containing 0.05% Tween 20 and a solution of clean peroxidase substrate solution (Kirkegaard & Perry, prepared according to the manufacturer's instructions). The reaction of the peroxidase with the substrate resulted in the formation of a dark green product, the intensity of the color being proportional to the amount of antibody present in the serum sample. The results were read after 15 minutes at a wavelength of 405 to 414 nanometers using an ELISA plate reader and recorded in Optical Density units (D.0).

The results are shown in Figures 1 (a) to (b) (antibody response in C3H / HEN), Figures 2 (a) and 2 (b) (antibody response in C57BL / 6) and Figures 3 (a ) and 3 (b) (antibody response in BALB / C). Inoculation of all three strains of C3H / HEN (H-2k), BALB / C (H-2d) and C3H / HEN (H-2b) mice with NS180RLfA9 antigen resulted in the formation of antibodies that reacted strongly with the region non-repetition of the circosporozoite protein. The reactivity to the NS1? RLfA9 capture antigen was only slightly greater than the reactivity to R16CSP. (The capture antigen R16CSP will only detect antibodies against a non-repeat portion of the CS protein whereas the NS1gRRff9 capture antigen allows the detection of both anti-NSl81 and anti-RLfA9 antibodies). As expected, C3H / HEN mice did not develop an antibody response to R32tetg2 whereas C57B1 / 6 mice developed. Although significantly weaker (1 log difference) than the antibody response observed in C57BL / 6 mice, the antibody response of BALB / C mice to R32tet82 was not expected and is contrary to the response

(89) SBC CASE 14445 reported in the literature for mice of this haplotype with respect to (NANP) 4q (Del Giudice, SLL, sL Immunol 137: 2952 (1986) reports that of fourteen strains of mice with nine haplotypes H- 2 mice, including BALB / C (H-2d), immunized with (NANP &gt; 4q, without a carrier protein, only H-mice developed an antibody response against (NANP) / C) mice did not respond in any way. BALB / C mice immunized with (FNK) coupled with keyhole limpet hemocyanin as the carrier protein did not develop anti- (NANP) 40 antibodies.

Inhibition of the Invasion of Escorozoite (IIE1

In this study, sera from rabbits immunized with NSlgβRLFA9 were tested for their ability to inhibit entry of faloaral sporozoites into liver cells, the site of development and maturation of sporozoites in the exo-erythrocytic phase.

Three mouse strains, BALB / C, C57BL / 6 and A / J, immunized with NSlβRLfA9, given either in complete Freund's adjuvant or in aluminum hydroxide, developed elevated antibody titers relative to the non-repeating region of the mouse. CS protein. However, sera from these mice were unable to block sporozoite invasion of cultured human hepatoma cells (HepG2-A16) when tested according to the Sporozoite Invasion Inhibition (IIE) assay described in Hollingdale, . al. sL. Immunol. 132 (7): 909 (1984).

In contrast, New Zealand white rabbits, immunized with μg NS1g RLfA9 administered in complete Freund's adjuvant at week 0.3 and 7, developed elevated antibody titers (though lower than those measured in mice) in relation to the non-repeat CS protein. However, sera from rabbits immunized with NSlgβRLÎ ± 9 significantly inhibited (98% in an animal, with the mean inhibition being about 60%) the invasion of hepatoma cells by

70 893 SBC CASE 14445 -36- sporozoites when tested according to the Hollingdale IIE assay.

Sporozoites of a chloroquine-resistant strain E. falciparum (7G8 strain) and E. falciparum strain (NF54 strain) sensitive to chloroquine were each significantly inhibited from entering hepatoma cells by rabbit sera immunized with NS1? RLfA9. Inhibition of hepatoma invasion by E. falciparum sporozoites of strain 7G-8 was superior (85% on average) to the ERI determined for strain NF54 (60% on average).

In IIE studies in which human hepatoma cells were replaced by normal human hepatocytes, E. falciparum sporozoites of NF54 strain were inhibited (89% inhibition by rabbit serum, with the mean inhibition being about 45%) to enter the hepatocytes by sera from rabbits immunized with NSl81RLfA9.

These studies suggest the presence of sporozoite neutralizing epitopes on the NSl81RLfA9 antigen.

Claims (30)

A method of preparing a polypeptide which can be used in a vaccine to protect humans against infection by Plasmodium, characterized in that it comprises (a) insertion of a coding sequence containing one or more immunogenic determinants of the first flanking region of a Plasmodium surface protein, one or more immunogenic determinants of the second flanking region, and containing none or containing less than all of the repeating immunogenic repeating domain determinants between them in a vector of expression in such a way that the coding sequence is operably linked to a regulatory element, (b) transforming a microorganism or cell to the vector, (c) culturing the microorganism or transformed cell such that the polypeptide is produced; and (d) collecting the polypeptide. A method according to claim 1, characterized in that the coding sequence comprises at least one immunogenic repeating determinant of a Plasmodium surface protein repeat domain between the first and second flanking regions. A method according to claim 1, characterized in that the coding sequence comprises substantially all of the immunogenic determinants of the first flanking region and the second flanking region. A method according to claim 1, characterized in that the immunogenic determinants of a Plasmodium surface protein are selected from the surface proteins of P. falciparum. P. vivax. P. malarial or P. ovale. A method according to claim 3, characterized in that the coding sequence further comprises at least one immunogenic determinant of a Plasmodium surface protein repeat domain between the first and second flanking regions. A method according to any one of claims 1 to 388, characterized in that the coding sequence is fused to a carrier protein. A method according to any one of claims 1 to 6, characterized in that the surface protein is a circumsporozoite (CS) protein. 8. A process according to claim 6, wherein the carrier protein comprises eighty-one N-terminal amino acids of the non-structural influenza virus 1 protein. A method according to claim 3, wherein the first flanking region comprises an amino acid sequence corresponding to amino acids 19 (Leu) to 123 (Pro) of the P. falciparum CS protein and in that the second flanking region comprises an amino acid sequence corresponding to amino acids 297 (Gly) to 412 (Asn) of the P. falciparum CS protein. A method according to claim 1, characterized in that the first flanking region comprises an amino acid sequence corresponding to amino acids 19 (Leu) to 123 (Pro) of the P. falciparum CS protein and in that the second flanking region comprises an amino acid sequence corresponding to amino acids 288 (Asn) to 412 (Asn) of the P. falciparum CS protein. A method according to any one of claims 9 and 10, characterized in that the coding sequence additionally comprises an immunogenic determinant of the formula - (Asn-XY-Pro) n -, in which X is Ala or Val, Y is Asn or Asp en is an integer less than 41 when the immunogenic determinant is positioned between the flanking regions and less than 100 when the immunogenic determinant precedes the flanking regions. 12. A process according to claim 1, wherein the coding sequence is of the formula NS1-Asp-His-Met-Leu-Thr-Asp-Pro-CSig_i23_cs297-412 A process according to claim 1, characterized in that the coding sequence is of the formula ## STR1 ## in which the coding sequence is of the formula: ## STR1 ## Asp-His-Met-Leu-Thr-Asp-Pro-CSi 9 -23-Gly -CS288-412- 14. A process according to claim 1, wherein the coding sequence is of the formula NSIII-Asp-His-Met-Leu-Thr-Asp-Pro-CSjg_223- (Asn-XY-Pro) n-Gly-CS288 -412 in which X is Ala or Val, Y is Asn or Asp en is an integer greater than or equal to one and less than 41. 15. A process according to claim 1, wherein the coding sequence is of the formula NSlg-Asp-His-Met-Leu-Thr-Asp-Pro-CSig_23- (Asn-Ala-Asn-Pro) n-Gly -CS288-412 wherein n is an integer greater than or equal to one and less than 41. A process according to claim 15, characterized in that n is equal to 2. 17. A process according to claim 1, wherein the coding sequence is of the formula NS1-Asp-His-Met-Leu-Thr-Asp-Pro-CSi9 [23] (Asn-Val-Asp-Pro) n-Gly -CS288-412 wherein n is an integer greater than or equal to one and less than 41. 18. A process as claimed in claim 1, wherein the coding sequence is of the formula NS1-181- (Asn-XY-Pro) n-Asp-His-Met-Leu-Thr-Asp-Pro-CSi9_23-Gly-CS288 -412 in n is an integer greater than or equal to one and less than 100. 19. A process according to claim 1, wherein the coding sequence is of the formula NSIIIgl (Asn-Ala-Asn-Pro) n-Asp-His-Met-Leu-Thr-Asp-Pro-CSi923-Gly -CS288-412 wherein n is an integer greater than or equal to one and less than 100. A process as claimed in claim 19, wherein n is equal to 4. A process according to claim 1, wherein the coding sequence is of the formula NS1-181 (Asn-Val-Asp-Pro) n-Asp-His-Met-Leu-Thr-Asp-Pro-CSi 9 23-Gly -CS288-412 wherein n is an integer greater than or equal to one and less than 100. -40- 70 893 SBC CASE 14445 A process according to claim 21, characterized in that n is equal to 4. A method according to claim 1, characterized in that the expression vector is an E. coli expression vector. A method according to claim 1, characterized in that the expression vector in E. coli is the pMG-1. 25. A process according to claim 1, characterized in that the expression vector in E. coli is pNSIIIRL19. A method according to claim 1, characterized in that the expression vector in E. coli is pNSlgγ RLfAuth- A process according to claim 1, characterized in that the expression vector in E. coli is NS1g RLfAuth ++ (NANP) n, where n is an integer greater than or equal to one. A method according to claim 1, characterized in that the expression vector in E. coli is pNSlgl (NANP) n-RLfAuth, where n is an integer greater than or equal to one. A method according to claim 1, characterized in that the expression vector in E. coli is pNSlg (NVDP) n RLfAuth, where n is an integer greater than or equal to one. A vaccine for the protection of humans against infection caused by Plasmodium sporozoites characterized in that the vaccine comprises mixing an immunoprotective amount of the polypeptides prepared according to claims 1 to 29 with a pharmaceutically acceptable carrier. Lisbon, &quot; 0. MAI 1990 By SMITHKLINE BEECHAM CORPORATION 0 AG! iFICIAL -